Human immunodeficiency virus (HIV), the etiological agent of acquired immunodeficiency syndrome (AIDS), enters cells by binding its envelope glycoprotein (Env) to receptor molecules and fusing its membrane with the cell membrane. Understanding how receptor molecules mediate HIV entry is critical for elucidating the mechanisms of HIV infection, and design of new antiretroviral treatments and vaccines. We have proposed to identify novel broadly HIV neutralizing human monoclonal antibodies (nhmAbs) by screening antibody phage display libraries (PDLs) against HIV envelope glycoproteins (Envs) complexed with receptor molecules. Last year we reported the identification of a novel hmAb Fab (X5) selected for binding to purified JRFL gp120-CD4-CCR5 complexes by screening of a PDL from an HIV-1-infected long-term nonprogressor, and preliminary data of its characterization. This year we have been extensively characterizing X5, published an article in PNAS (attached), and NCI filed a patent application and is negotiating license agreements with three companies. Fab X5 bound with high (nM) affinity to a variety of Envs including primary isolates from different clades and Envs with deleted variable loops (V1,2,3). Its binding was significantly increased by CD4. X5 inhibited infection of peripheral blood mononuclear cells by a selection of representative HIV-1 primary isolates from clades A, B, C, D and G with an efficiency comparable to that of the broadly neutralizing antibody IgG1 b12. Furthermore X5 inhibited cell fusion mediated by Envs from R5, X4 and R5X4 viruses. Of the five broadly cross-reactive HIV nhmAbs X5 is the only one that exhibits increased binding to gp120 complexed with receptors. Our collaborator, Dr. Ji, and his associates have solved the crystal structure of X5 at 1.9 A resolution, and we are currently characterizing the structure of its epitope by site-directed mutagenesis and molecular docking of X5 to known structures of gp120 cores. We also developed a novel methodology based on alternating various Envs (gp140s) and their complexes with CD4 during panning of PDLs. By using this approach we selected four new potent broadly HIV nhmAb Fabs (M12,14,16,18) from a PDL that is different than the one used for selection of X5. Preliminary data suggest that these antibodies potently inhibit cell-cell fusion mediated by Envs of primary isolates from different clades. NCI filed a patent application for these antibodies. These findings suggest that X5, M12, M14, M16 and M18 could be possibly used as entry inhibitors alone or in combination with other antiretroviral drugs for the treatment of HIV-infected individuals, provide evidence for the existence of conserved receptor-induced gp120 epitopes that can serve as targets for potent broadly cross-reactive neutralizing antibodies in HIV-infected patients, and have important conceptual and practical implications for development of vaccines and inhibitors. The HIV-1 envelope glycoprotein (Env, gp120-gp41) undergoes a series of conformational changes initiated by its binding to receptor molecules. I hypothesized that some of the transient Env conformations on the pathway to entry can be exhibited and retained in fusion proteins of gp120 and gp41 joined by flexible linkers. Two Env89.6 fusion proteins with linkers of 14 and 26 amino acid residues inhibited 10-100-fold more potently entry and cell fusion mediated by Envs from R5 (ADA, JRFL, SF162, Bal), X4 (HXB2, NL4-3) and R5X4 (89.6, DH12) HIV-1 isolates than a fusion protein with a 4 amino acid residue linker obtained under the same conditions, gp120-gp41 without linker or gp120. The high inhibitory activity (50% at about 100 pM - higher than of any currently known HIV-1 entry inhibitor on molar basis) of these proteins against a variety of HIV-1 isolates, including primary isolates, may suggest the existence of broadly conserved structures that are critical for the entry process and can be exposed even in the absence of receptor-mediated activation thus opening new perspectives for elucidation of viral entry mechanisms, and development of inhibitors and vaccines.